Functional analysis of FMRP is hampered due to a lack of functional assays. In this project we propose the development of two distinct assays for FMRP function. The first system is based upon our recent discovery that FMRP can transform NIH 3T3 cells. This is consistent with the biochemical attributes of FMRP as another translation factor, eIF-4E, has previously been shown to cause transformation. This system will be developed as an assay for FMRP function. We will determine if RNA-binding and/or nucleocytoplasmic shuttling is required for transformation. We will seek to understand how excess FMRP causes transformation as this may lead to more general insight into function. As additional attributes of FMRP become known, we will test them in this system as well. The second system is based upon the fmr1 knockout mouse which exhibits cognitive deficit. In order to answer fundamental questions regarding fragile X syndrome and to provide a more experimentally useful murine knockout model, we will replace, in ES cells, the normal mouse fmr1 promoter with the tetracycline-response element promoter without other wise modifying the murine gene. Simultaneously, we will insert the rtetR/VP16 fusion transcription factor, under the control of the normal Fmr1 promoter, into the X-linked HPRT locus to achieve single copy integration. In these and derivative cell lines, addition of the tetracycline derivative, doxycycline, will result in turning the Fmr1 gene on. Mice will be developed from these ES cells, resulting in animals in which both temporal and quantitative control over FMRP expression can be controlled by doxycycline exposure. We will then determine if FMRP expression is essential during development or if later expression of FMRP can influence the cognitive deficit. Such data is of fundamental importance in the consideration of therapeutic interventions. Developing, testing, and utilizing these model system should allow considerable analysis and understanding of FMRP function and the consequences of its absence.